Folding Landscape Research Articles

Thermodynamic Basis for the Stabilization of Helical Peptoids by Chiral Sidechains.

Peptoids are a class of highly customizable biomimetic foldamers that retain properties from both proteins and polymers. It has been shown that peptoids can adopt peptide-like secondary structures through the careful selection of sidechain chemistries, but the underlying conformational landscapes that drive these assemblies at the molecular level remain poorly understood. Given the high flexibility of the peptoid backbone, it is essential that methods applied to study peptoid secondary structure formation possess the requisite sensitivity to discriminate between structurally similar yet energetically distinct microstates. In this work, a generalizable simulation scheme is used to robustly sample the complex folding landscape of various 12mer polypeptoids, resulting in a predictive model that links sidechain chemistry with preferential assembly into one of 12 accessible backbone motifs. Using a variant of the metadynamics sampling method, four peptoid dodecamers are simulated in water: sarcosine, N-(1-phenylmethyl)glycine (Npm), (S)-N-(1-phenylethyl)glycine (Nspe), and (R)-N-(1-phenylethyl)glycine (Nrpe)─to determine the underlying entropic and energetic impacts of hydrophobic and chiral peptoid sidechains on secondary structure formation. Our results indicate that the driving forces to assemble Nrpe and Nspe sequences into polyproline type-I helices in water are found to be enthalpically driven, with small benefits from an entropic gain for isomerization and steric strain due to the presence of the chiral center. The minor entropic gains from bulky chiral sidechains in Nrpe- and Nspe-containing peptoids can be explained through increased configurational entropy in the cis state. However, overall assembly into a helix is found to be overall entropically unfavorable. These results highlight the importance of considering the many various competing interactions in the rational design of peptoid secondary structure building blocks.

RNA folding pathways from all-atom simulations with a variationally improved history-dependent bias

Atomically detailed simulations of RNA folding have proven very challenging in view of the difficulties of developing realistic force fields and the intrinsic computational complexity of sampling rare conformational transitions. As a step forward in tackling these issues, we extend to RNA an enhanced path-sampling method previously successfully applied to proteins. In this scheme, the information about the RNA’s native structure is harnessed by a soft history-dependent biasing force promoting the generation of productive folding trajectories in an all-atom force field with explicit solvent. A rigorous variational principle is then applied to minimize the effect of the bias. Here, we report on an application of this method to RNA molecules from 20 to 47 nucleotides long and increasing topological complexity. By comparison with analog simulations performed on small proteins with similar size and architecture, we show that the RNA folding landscape is significantly more frustrated, even for relatively small chains with a simple topology. The predicted RNA folding mechanisms are found to be consistent with the available experiments and some of the existing coarse-grained models. Due to its computational performance, this scheme provides a promising platform to efficiently gather atomistic RNA folding trajectories, thus retain the information about the chemical composition of the sequence.

Odd–even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation

Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid-liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA-RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum-which depends on the exact sequence, n, and ionic conditions-is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

Sequential Unfolding Mechanisms of Monomeric Caspases.

Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3-10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∼15 kcal mol-1, near the physiological pH range (pH 6-8) and unfold via two partially folded intermediates, I1 and I2*, that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.

Is Posttranslational Folding More Efficient Than Refolding from a Denatured State: A Computational Study.

The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular machine responsible for protein synthesis, is known to interact with nascent proteins, adding further complexity to the protein folding landscape. Consequently, it is unclear whether the folding pathways of proteins are conserved on and off the ribosome. The main question remains: to what extent does the ribosome help proteins fold? To address this question, we used coarse-grained molecular dynamics simulations to compare the mechanisms by which the proteins dihydrofolate reductase, type III chloramphenicol acetyltransferase, and d-alanine-d-alanine ligase B fold during and after vectorial synthesis on the ribosome to folding from the full-length unfolded state in bulk solution. Our results reveal that the influence of the ribosome on protein folding mechanisms varies depending on the size and complexity of the protein. Specifically, for a small protein with a simple fold, the ribosome facilitates efficient folding by helping the nascent protein avoid misfolded conformations. However, for larger and more complex proteins, the ribosome does not promote folding and may contribute to the formation of intermediate misfolded states cotranslationally. These misfolded states persist posttranslationally and do not convert to the native state during the 6 μs runtime of our coarse-grain simulations. Overall, our study highlights the complex interplay between the ribosome and protein folding and provides insight into the mechanisms of protein folding on and off the ribosome.

Probing the Protein Folding Energy Landscape: Dissociation of Amyloid-β Fibrils by Laser-Induced Plasmonic Heating.

Insoluble amyloid fibrils made from proteins and peptides are difficult to be degraded in both living and artificial systems. The importance of studying their physical stability lies primarily with their association with human neurodegenerative diseases, but also owing to their potential role in multiple bio-nanomaterial applications. Here, gold nanorods (AuNRs) were utilized to investigate the plasmonic heating properties and dissociation of amyloid-β fibrils formed by different peptide fragments (Aβ16-22/Aβ25-35/Aβ1-42) related to the Alzheimer's disease. It is demonstrated that AuNRs were able to break mature amyloid-β fibrils from both the full length (Aβ1-42) and peptide fragments (Aβ16-22/Aβ25-35) within minutes by triggering ultrahigh localized surface plasmon resonance (LSPR) heating. The LSPR energy absorbed by the amyloids to unfold and move to higher levels in the protein folding energy landscape can be measured directly and in situ by luminescence thermometry using lanthanide-based upconverting nanoparticles. We also show that Aβ16-22 fibrils, with the largest persistence length, displayed the highest resistance to breakage, resulting in a transition from rigid fibrils to short flexible fibrils. These findings are consistent with molecular dynamics simulations indicating that Aβ16-22 fibrils possess the highest thermostability due to their highly ordered hydrogen bond networks and antiparallel β-sheet orientation, hence affected by an LSPR-induced remodeling rather than melting. The present results introduce original strategies for disassembling amyloid fibrils noninvasively in liquid environment; they also introduce a methodology to probe the positioning of amyloids on the protein folding and aggregation energy landscape via nanoparticle-enabled plasmonic and upconversion nanothermometry.

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.

Reframing prosegment-dependent folding and limits on natural protein folding landscapes from an evolutionary perspective.

In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha-lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self-assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non-cooperativity, memory effects, and substantial kinetic trapping. The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding "exceptions"-including protein fold switching, functional misfolding, and prevalent inability to refold-suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.

Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1-40.

Amyloid fibrils represent the structural endpoint on the energetic (mis)folding landscape of very many proteins. Physiologically, amyloid fibrils are observed as a characteristic hallmark in misfolding diseases often associated with degenerative and neurodegenerative disorders. In the beginning of the scientific discussion, the focus is laid on the fibrillar state, but over the time it becomes increasingly clear that low molecular weight and transient aggregates are of crucial importance for pathological mechanisms. Structural studies find different intra- and intermolecular contacts for the most well-studied peptide amyloid β (Aβ) depending on the stage of fibrillation. In particular, the contact between residues phenylalanine 19 (F19) and leucine 34 (L34) seems to be highly conserved, suggesting that it must be of particular significance for Aβ misfolding and possibly the pathological properties of the peptide. This review aims to highlight the rational and the usefulness of point mutations in Aβ peptides and their impact on the critical interstrand contact F19-L34 depending on the stage of fibrillation. While the amyloid structure of Aβ is very robust against quite a few modifications, the toxicity of mutated Aβ molecules highly depends on the F19-L34 contact.

Conserved folding landscape of monomeric initiator caspases

The apoptotic caspase subfamily evolved into two subfamilies-monomeric initiators and dimeric effectors; both subfamilies share a conserved caspase-hemoglobinase fold with a protease domain containing a large subunit and a small subunit. Sequence variations in the conserved caspase-hemoglobinase fold resulted in changes in oligomerization, enzyme specificity, and regulation, making caspases an excellent model for examining the mechanisms of molecular evolution in fine-tuning structure, function, and allosteric regulation. We examined the urea-induced equilibrium folding/unfolding of two initiator caspases, monomeric caspase-8 and cFLIPL, over a broad pH range. Both proteins unfold by a three-state equilibrium mechanism that includes a partially folded intermediate. In addition, both proteins undergo a conserved pH-dependent conformational change that is controlled by an evolutionarily conserved mechanism. We show that the conformational free energy landscape of the caspase monomer is conserved in the monomeric and dimeric subfamilies. Molecular dynamics simulations in the presence or the absence of urea, coupled with limited trypsin proteolysis and mass spectrometry, show that the small subunit is unstable in the protomer and unfolds prior to the large subunit. In addition, the unfolding of helix 2 in the large subunit results in disruption of a conserved allosteric site. Because the small subunit forms the interface for dimerization, our results highlight an important driving force for the evolution of the dimeric caspase subfamily through stabilizing the small subunit.

I-Motif folding intermediates with zero-nucleotide loops are trapped by 2′-fluoroarabinocytidine via F···H and O···H hydrogen bonds

G-quadruplex and i-motif nucleic acid structures are believed to fold through kinetic partitioning mechanisms. Such mechanisms explain the structural heterogeneity of G-quadruplex metastable intermediates which have been extensively reported. On the other hand, i-motif folding is regarded as predictable, and research on alternative i-motif folds is limited. While TC5 normally folds into a stable tetrameric i-motif in solution, we report that 2′-deoxy-2′-fluoroarabinocytidine (araF-C) substitutions can prompt TC5 to form an off-pathway and kinetically-trapped dimeric i-motif, thereby expanding the scope of i-motif folding landscapes. This i-motif is formed by two strands, associated head-to-head, and featuring zero-nucleotide loops which have not been previously observed. Through spectroscopic and computational analyses, we also establish that the dimeric i-motif is stabilized by fluorine and non-fluorine hydrogen bonds, thereby explaining the superlative stability of araF-C modified i-motifs. Comparative experimental findings suggest that the strength of these interactions depends on the flexible sugar pucker adopted by the araF-C residue. Overall, the findings reported here provide a new role for i-motifs in nanotechnology and also pose the question of whether unprecedented i-motif folds may exist in vivo.

Inferring Pathways of Oxidative Folding from Prefolding Free Energy Landscapes of Disulfide-Rich Toxins.

Short, cysteine-rich peptides can exist in stable or metastable structural ensembles due to the number of possible patterns of formation of their disulfide bonds. One interesting subset of this peptide group is the conotoxins, which are produced by aquatic snails in the family Conidae. The μ conotoxins, which are antagonists and blockers of the voltage-gated sodium channel, exist in a folding spectrum: on one end of the spectrum are more hirudin-like folders, which form disulfide bonds and then reshuffle them, leading to an ensemble of kinetically trapped isomers, and on the other end are more BPTI-like folders, which form the native disulfide bonds one by one in a particular order, leading to a preponderance of conformations existing in a single stable state. In this Article, we employ the composite diffusion map approach to study the unified free energy surface of prefolding μ-conotoxin equilibrium. We identify the two most important nonlinear collective modes of the unified folding landscape and demonstrate that in the absence of their disulfides, the conotoxins can be thought of as largely disordered polymers. A small increase in the number of hydrophobic residues in the protein shifts the free energy landscape toward hydrophobically collapsed coil conformations responsible for cysteine proximity in hirudin-like folders, compared to semiextended coil conformations with more distal cysteines in BPTI-like folders. Overall, this work sheds important light on the folding processes and free energy landscapes of cysteine-rich peptides and demonstrates the extent to which sequence and length contribute to these landscapes.

Folding and threading of leptin as a model for pierced lasso topologies.

The introduction of entanglements in proteins remains elusive, where the mechanism of formation is unclear. We have for the first time characterized the folding and threading mechanism of a “knot-like” protein. The recently discovered Pierced Lasso Topology (PLT) is a class of proteins in which a disulfide formed covalent loop is threaded by the protein backbone of a free terminus. Within the last decade PLTs have been identified in over 600 known protein structures across all kingdoms of life. These proteins vary in function from oxidoreductases to hydrolases and small hormones. Their presence in different cellular compartments is highly influenced by the redox potential of the environment acting as a potential molecular switch. A combination of experimental phi-value analysis and structure-based models were used to identify key residues within the folding pathway for the model human hormone leptin. Previous computational work uncovered that the threading event occurs within the early stage of the folding free energy landscape. Incorporation of a threading event adds ruggedness to the folding landscape, slowing the process. Our investigation of leptin's in vitro threading event utilized a variant designed to lock the structure in a pre-threaded conformation inhibiting unthreading of the chain in the denatured state. The designed variant folds 100 times faster than the wild type, demonstrating that leptin spontaneously threads/unthreads in oxidizing environments; with the covalent loop intact.

An evaluation of force field accuracy for the mini-protein chignolin using Markov state models.

All-atom molecular dynamics (MD) simulations can provide detailed insight into a molecule's conformational ensemble in solution. While molecular force fields are parameterized to accurately model a protein's potential energy surface, it remains challenging in practice to evaluate how well force fields can capture ensemble-averaged experimental observables, since it requires simulation of the complete folding landscape. In this work, we employ massively parallel molecular simulations, performed using the [email protected] distributed computing platform, to investigate the ability of nine force fields (AMBER14SB, AMBER99, AMBER99SB, AMBER99SB-ildn, AMBER99SBnmr1-ildn, CHARMM22∗, CHARMM27, CHARMM36 and OPLS-aa) with TIP3P explicit solvent to accurately reproduce experimental observables for chignolin, a beta-hairpin mini-protein with an experimental folding time of ∼600 ns. From over 200 microseconds of aggregate trajectory data, we constructed Markov state models (MSMs) to obtain estimates of thermodynamic and kinetic properties of chignolin in each force field. Quantitative assessment of the force fields was performed by comparing predicted and experimental folded populations, and the statistical agreement between predicted and experimental solution-state NMR observables. Our results rank force field accuracy for chignolin similarly compared to previous work using oligopeptides and ubiquitin (Beauchamp et al. 2012). This work highlights the utility of MSM approaches for force field evaluation, and provides a baseline for future studies using Bayesian inference methods to evaluate and parameterize force fields. It also provides an excellent dataset for testing new approaches to simulation-based sequence design of mini-proteins.

Examining the importance of termini location in successful protein folding with HaloTag circular permutants in vitro, in E. coli, and in mammalian cells.

Though in vitro studies have produced insight into mechanisms underlying protein folding, determining how cellular factors modulate folding remains challenging. Proteins have the opportunity to fold when synthesized from N- to C-terminus by the ribosome at a rate of around five to ten residues per second in cells. This is much slower than the average folding rate of most small proteins, giving the nascent chain time to sample many conformations during translation. When refolding in vitro, HaloTag populates an aggregation-prone intermediate that contains structure from both the N- and C-termini, but this intermediate does not form co-translationally. Could altering the chain connectivity of HaloTag decrease its folding efficiency? We have shown how changing the vectorial synthesis of HaloTag through circular permutation affects folding efficiency in vitro and in vivo. We assessed all 298 circular permutants of HaloTag in E. coli cell lysate and showed that circular permutation positions differentially affect protein solubility and function. FACS experiments in live mammalian HEK293T cells demonstrate that many circular permutants retain function in an alternative cellular environment. Combining these data with both standard in vitro protein folding assays and hydrogen-exchange MS studies to map the folding process yields insight into how circular permutation alters the folding landscape of HaloTag and the role of co-translational folding in this process.

Creative destruction: New protein folds from old

Mechanisms of emergence and divergence of protein folds pose central questions in biological sciences. Incremental mutation and stepwise adaptation explain relationships between topologically similar protein folds. However, the universe of folds is diverse and riotous, suggesting more potent and creative forces are at play. Sequence and structure similarity are observed between distinct folds, indicating that proteins with distinct folds may share common ancestry. We found evidence of common ancestry between three distinct β-barrel folds: Scr kinase family homology (SH3), oligonucleotide/oligosaccharide-binding (OB), and cradle loop barrel (CLB). The data suggest a mechanism of fold evolution that interconverts SH3, OB, and CLB. This mechanism, which we call creative destruction, can be generalized to explain many examples of fold evolution including circular permutation. In creative destruction, an open reading frame duplicates or otherwise merges with another to produce a fused polypeptide. A merger forces two ancestral domains into a new sequence and spatial context. The fused polypeptide can explore folding landscapes that are inaccessible to either of the independent ancestral domains. However, the folding landscapes of the fused polypeptide are not fully independent of those of the ancestral domains. Creative destruction is thus partially conservative; a daughter fold inherits some motifs from ancestral folds. After merger and refolding, adaptive processes such as mutation and loss of extraneous segments optimize the new daughter fold. This model has application in disease states characterized by genetic instability. Fused proteins observed in cancer cells are likely to experience remodeled folding landscapes and realize altered folds, conferring new or altered functions.

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants.

The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.

The Non-native Disulfide-Bond-Containing Landscape Orthogonal to the Oxidative Protein-Folding Trajectory: A Necessary Evil?

Oxidative protein folding describes the process by which disulfide-bond-containing proteins mature from their ribosomal, fully reduced and unfolded, origins. Over the past 40 years, a number of exemplar proteins including bovine pancreatic ribonuclease A (RNaseA), bovine pancreatic trypsin inhibitor (BPTI), and hen egg-white lysozyme (HEWL), among others, have provided rich insight into the nature of the intermolecular interactions that drive the formation of the native, biologically active fold. In this Review Article, we revisit the oxidative folding process of RNaseA with a focus on reconciling the role of non-native disulfide-bond-containing species that populate the oxidative folding landscape. Toward gaining such an understanding, we project the regeneration pathway onto a Cartesian coordinate system. This helps not only to recognize the magnitude of the seemingly "fruitless", non-native disulfide-bond-containing species that lie orthogonal to the "native-protein-forming" reaction progress but also to reconcile a role for their existence in the regenerative trajectory. Finally, we superimpose the folding funnel onto the regeneration trajectory to draw parallels between oxidative folders and conformational folders (proteins that lack disulfide bonds). The overall objective is to provide the reader with a semi-quantitative description of oxidative protein folding and the barriers to successful regeneration while underscoring a role of seemingly fruitless intermediates.

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.